Radiation induced non-targeted/bystander effects have been demonstrated with a variety of biological endpoints using mammalian cell cultures, 3D human tissues, and more recently, in C. elegans and in mice. However, neither the mechanism nor the relevance of the bystander response to human health is clear. This program project brings together and links 3 highly integrated projects aimed at shedding new light on the mechanism and health relevance of the non-targeted/bystander phenomenon using both in vitro and in vivo approaches. The overall hypothesis of this program project is that the radiation induced non-targeted (bystander) response can be initiated both in vitro and in vivo by oxidative stress and propagated by multifactorial signaling events involving cyclooxygenase-2 (COX-2) and junctional communication, and is modulated by the status of the Rad9 protein. The three projects are highly interactive in goals and research approaches and are further linked together by a technical core, which will provide specialized irradiation facilities, data analyses and state-of-the-art gene expression profile related technologies. Project 1 will address the role of Rad9, a DNA damage response protein, in the radiation induced bystander effect in mouse embryo fibroblasts (MEF) and mice. The underlying hypothesis to be tested is that Rad9 controls the bystander process via regulation of COX-2, p21Waf1 and other downstream targets. Project 2 will build on the preliminary findings that radiation induces non-targeted, out of field mutagenesis in lung tissues of gpt delta transgenic mice to test the hypothesis that COX-2 mediates radiation-induced bystander mutagenesis in vivo and that the bystander cells are genomically unstable in genetically susceptible populations. Project 3 will address the central hypothesis that the in vivo cellular microenvironment modulates gap junction gating that determines the nature of signaling events propagated between directly-irradiated and bystander cells. The projects are conceptually linked and technically interactive such that they complement cross-talk and strengthen each other. The observation that the progeny of non-targeted cells show an increase in genomic instability as evidenced by an increase in delayed mutations and chromosomal aberrations many generations post-irradiation indicate the need for a comprehensive assessment of the bystander issue, particularly among genetically susceptible population, to understand mechanism(s) and impact on human health.